1. Field of the Invention
The invention provides nucleotide sequences from coryneform bacteria which code for the metY gene and a process for the fermentative preparation of amino acids, in particular L-lysine and L-methionine, using bacteria in which at least the metY gene is enhanced.
2. Description of the Related Art
L-Amino acids, in particular L-lysine and L-methionine, are used in human medicine and in the pharmaceuticals of industry, in the foodstuffs industry and very particularly in animal nutrition.
It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation process. Improvements to the process can relate to fermentation measures stirring and supply of oxygen, or to the composition of the nutrient media, such as the sugar concentration during the fermentation, or to working up of the product by, for example, ion exchange chormatography, of to the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and produce amino acids are obtained in this manner.
Recombinant DNA techniques have also been employed for some years for improving the Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating their effect on amino acid production.
An object of the present invention is to provide new measures for improved fermentative preparation of amino acids, in particular L-lysine and L-methionine.
Where L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.
When L-lysine or lysine are mentioned in the following, not only the bases but also the salts, such as lysine monohydrochloride or lysine sulfate, are intended.
When L-methionine or methionine are mentioned in the following, the salts, such as e.g. methionine hydrochloride or methionine sulfate, are intended.
The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the metY gene, chosen from the group consisting of
a) polynucleotide which is at least 70% identical to a polynucleotide that codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
b) polynucleotide which codes for a polypeptide that comprises an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID No. 2,
c) polynucleotide which is complementary to the polynucleotides of a) or b), and
d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c),
and the corresponding polypeptides having the enzymatic activity of O-acetylhomoserine sulfhydrylase.
The invention also provides the above-mentioned polynucleotides as DNA which is capable of replication, comprising:
(i) the nucleotide sequence shown in SEQ ID No. 1, or
(ii) at least one sequence which corresponds to sequence
(i) within the range of the degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and optionally
(iv) sense mutations of neutral function in (i).
The invention also provides
a polynucleotide comprising the nucleotide sequence as shown in SEQ ID No. 1;
a polynucleotide that codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2;
a vector containing the DNA sequence of C. glutamicum that codes for the metY gene, deposited in accordance with the Budapest Treaty in Corynebacterium glutamicum as pCREmetY on Jun. 06, 2000 under DSM 13556
and coryneform bacteria in which the metY gene is present in enhanced form, in particular by the vector pCREmetY.
The invention also provides polynucleotides which are obtained by screening a corresponding gene library of a coryneform bacterium, which comprises the complete gene or parts thereof, by means of hybridization with a probe which comprises the sequence of the polynucleotide according to the invention according to SEQ ID No.1 or a fragment thereof, and isolation of the polynucleotide sequence mentioned.